Myocardial infarction is a leading cause of morbidity with loss of contractile performance precipitated by the limited capacity of heart muscle for self-renewal. The aptitude of embryonic stem cells for differentiation into cardiomyocytes provides an opportunity for regenerative repair. Indeed, the basis for therapeutic use of these quintessential stem cells lies in their inherent pluripotency, yet the distinct ability for multi-lineage differentiation carries the liability of neoplastic growth that outweighs benefit impeding reparative applications. The objective of this proposal is to hone stem cell plasticity in order to nullify malignancy-prone unguided differentiation for safe repair. Preliminary studies demonstrate the aptitude for definitive cardiogenic commitment of an embryonic stem cell population within the developing embryoid body, during a transient window of endoderm-directed cardiotrophic signaling, with the resulting specification of the mesoderm yielding a progenitor "cardiopoietic" stem cell phenotype. Aim #1 will (i) isolate this pro-cardiogenic population utilizing embryoid body-based stem cell differentiation, (ii)define its molecular identity, and (iii)determine its propensity for terminal cardiomyogenic transformation. Aim #2 will (i) decipher the endoderm-derived cardiotrophic signaling, (ii) recruit in an embryoid body-free manner the pro-cardiogenic cardiopoietic population at large scale, and (iii) validate in vivo the safety profile of this novel stem cell type. Aim #3 will (i) assess the curative potential of procured cardiopoietic stem cells in treating myocardial infarction,(ii) determine the underlying mechanism for repair, and (iii) engineer stem cells for augmented tolerance to stress achieving enhanced cardioprotective outcome. Based on the characterization of cardiopoietic stem cells and the profiling of cardiotrophic cues, this proposal will dissect the fundamental process of cardiopoiesis securing formative confinement of pluripotency. Dissection of cardiogenesis at the molecular and cellular level will be further integrated into a therapeutic paradigm, and translated to the organ and organism level for safe cell-based regenerative therapy of myocardial infarction.